1. Field of the Invention
The present invention relates generally to the fields of chemical analysis and biomedical diagnostics, and more particularly, to the use of fluorescence and absorption associated with the Stokes shift to determine the molecular components.
2. Description of the Related Art
The emission and absorption of organic molecules in different hosts has been studied since Newton's work on color and dispersion. Information on the state of matter has been obtained by measuring the emission and absorption spectra. For over four decades, spectroscopy has played an important role in understanding the fundamental processes that occur in biology, chemistry, and solid-state physics. In the context of biology, the presence of intrinsic fluorophores such as tryptophan, collagen, elastin, NADH, and flavins in animal and human tissues offer potential opportunities to probe the morphological changes occurring in diseased tissues using cw and time-resolved fluorescence spectroscopy (1).
Since 1984, fluorescence spectroscopy has been used as diagnostic tool to separate cancer, benign and surrounding tissue regions (2-5), Typically, a tissue is excited at a given wavelength to emit radiation at different wavelengths, which characterize the tissue. The wavelengths from UV to blue range (280–520 nm) excite proteins in the tissue associated with changes in structure and molecular content, which gives their spectral fingerprints. The particles in the tissue include tryptophan, collagen, elastin, and NADH.
The underlying dynamics of organic biomolecules occur on the energy configuration coordinate (E-Q) diagram (6-8), where E≡energy and Q≡normalized dimensionless lattice displacement coordinate. The electronic, vibration and rotational states associated with ground and excited states of a biomolecule are denoted on the E-Q diagram. The absorption and emission transition occurs as vertical transition between the states on the E-Q diagram (see FIG. 1). The time evolution of the process can be followed on E-Q diagram during nonradiative lattice relaxation and excitation (1). The ground and excited states are located at different equilibrium Q coordinate positions due to difference in electronic-lattice coupling. The difference in electronic-lattice coupling is denoted by the Huang-Rhys parameters, S.
Typically, the peaks of the absorption and the emission occur at different wavelengths. The emission band occurs at lower energy than the excitation band. The shift of the emission and absorption peaks is known as the Stokes shift given by □2Shω. The Stokes shift depends on the polarization of the host environment surrounding the emitting organic molecule. Large shifts are observed for a polar surrounding, such as water. There is dynamic change in the location of energy states associated with this interaction between dipole moment of transition and the surrounding host molecules during excitation and emission (8) 